Heparin-binding cationic peptide self-assembling peptide amphiphiles useful against drug-resistant bacteria

ABSTRACT

Disclosed are peptides comprising an amphiphilic backbone and a cationic heparin-binding motif peptide. The peptides can be used in methods of antimicrobial treatment.

RELATED APPLICATION

This application claims the benefit of priority to U.S. ProvisionalApplication for Patent Ser. No. 62/430,906, filed Dec. 6, 2016.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Feb. 23, 2018, isnamed NEX-02701 SL.txt and is 6,584 bytes in size.

BACKGROUND

Bacterial antibiotic resistance has become a critical threat to globalhealth and the worldwide economy. According to the Center for DiseaseControl and Prevention (CDC), over 2 million cases of illness, and23,000 deaths, are caused each year by bacterial drug-resistance in theUnited States [1]. Moreover, bacteria can develop resistance to a newantibiotic within a few years. Therefore, rather than continuallydeveloping new antibiotics that may be ineffective within a short time,there remains a need to develop novel antibacterial agents that target abroad range of bacteria without inducing bacterial drug-resistance.

SUMMARY

In one aspect, the disclosure provides a polypeptide comprising anamphiphilic backbone and a cationic heparin-binding motif peptide.

In another aspect, the disclosure provides a method of antimicrobialtreatment, comprising:

providing a sample comprising a plurality of microorganisms;

adding to the sample a polypeptide comprising an amphiphilic backboneand a cationic heparin-binding motif peptide;

thereby killing or inhibiting the growth of at least a portion of theplurality of microorganisms in the sample.

In another aspect, the disclosure provides a method of preventing orsuppressing microbial growth on a surface, comprising:

applying to the surface a polypeptide comprising an amphiphilic backboneand a cationic heparin-binding motif peptide;

thereby preventing or suppressing microbial growth on the surface.

In another aspect, the disclosure provides a method of preparingself-assembled nanorods comprising the steps of:

dissolving a plurality of lyophilized polypeptides comprising anamphiphilic backbone and a cationic heparin-binding motif peptide in asolvent to form a mixture;

mixing the mixture; and

storing the mixture to allow for supramolecular self-assembly,

thereby preparing self-assembled nanorods.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A depicts a schematic illustration of a molecular structure of anexemplary peptide, ACA-PA.

FIG. 1B depicts a schematic illustration of the self-assembly process ofan exemplary peptide, ACA-PA, into cylindrical structures in aqueoussolution.

FIG. 2A depicts a transmission electron microscope (TEM) image of theself-assembled morphology of a peptide, CVK-PA.

FIG. 2B depicts a TEM image of the self-assembled morphology of anexemplary peptide, ACA-PA, at 1 mg/mL.

FIG. 2C depicts a TEM image of the self-assembled morphology of anexemplary peptide, ACA-PA, at 2 mg/mL.

FIG. 3A depicts circular dichroism (CD) spectra showing the secondarystructure of exemplary peptides at concentrations of 0.4 mM in water.

FIG. 3B depicts CD spectra showing the secondary structure of exemplarypeptides at concentrations of 0.3 mM in prokaryotic mimicking liposomevesicles.

FIG. 3C depicts CD spectra showing the secondary structure of exemplarypeptides in 25 mM SDS solution.

FIG. 4A depicts fluorescence intensity of the Nile Red dye (Ex=550 nm)against log₁₀ values of the peptide concentration.

FIG. 4B depicts fluorescence intensity of the Nile Red dye at differentconcentrations of an exemplary peptide, ACA-PA.

FIG. 4C depicts fluorescence intensity of the Nile Red dye at differentconcentrations of a peptide, CVK-PA.

FIG. 4D depicts fluorescence intensity of the Nile Red dye at differentconcentrations of Bi-Cardin peptide.

FIG. 5A depicts bacterial growth inhibition induced by an exemplarypeptide, ACA-PA, against Gram-positive MRSA.

FIG. 5B depicts bacterial growth inhibition induced by an exemplarypeptide, ACA-PA, against Gram-positive S. aureus.

FIG. 5C depicts bacterial growth inhibition induced by an exemplarypeptide, ACA-PA, against Gram-negative MDR E. coli.

FIG. 5D depicts bacterial growth inhibition induced by an exemplarypeptide, ACA-PA, against Gram-negative E. coli.

FIG. 6A depicts bacterial growth inhibition induced by Bi-Cardin againstGram-positive MRSA.

FIG. 6B depicts bacterial growth inhibition induced by Bi-Cardin againstGram-positive S. aureus.

FIG. 6C depicts bacterial growth inhibition induced by Bi-Cardin againstGram-negative MDR E. coli.

FIG. 6D depicts bacterial growth inhibition induced by Bi-Cardin againstGram-negative E. coli.

FIG. 7A depicts bacterial growth inhibition induced by a peptide,CVK-PA, against Gram-positive MRSA.

FIG. 7B depicts bacterial growth inhibition induced by a peptide,CVK-PA, against Gram-positive S. aureus.

FIG. 7C depicts bacterial growth inhibition induced by a peptide,CVK-PA, against Gram-negative MDR E. coli.

FIG. 7D depicts bacterial growth inhibition induced by a peptide,CVK-PA, against Gram-negative E. coli.

FIG. 8A depicts the bactericidal effect of an exemplary peptide, ACA-PA,against Gram-positive bacteria.

FIG. 8B depicts the bactericidal effect of an exemplary peptide, ACA-PA,against Gram-negative bacteria.

FIG. 9A depicts a TEM image of the self-assembled morphology of anexemplary peptide, ACA-PA, after mixing with heparin.

FIG. 9B depicts the bactericidal effect of an exemplary peptide, ACA-PA,against drug-resistant bacteria after mixing with heparin in variousweight ratios.

FIG. 10A depicts bacterial viability using a live/dead assay foruntreated Gram-positive MRSA.

FIG. 10B depicts bacterial viability using a live/dead assay forGram-positive MRSA with 40 μM of an exemplary peptide, ACA-PA.

FIG. 10C depicts bacterial viability using a live/dead assay forGram-positive MRSA with 80 μM of an exemplary peptide, ACA-PA.

FIG. 10D depicts bacterial viability using a live/dead assay foruntreated Gram-negative MDR E. coli.

FIG. 10E depicts bacterial viability using a live/dead assay forGram-negative MDR E. coli with 40 μM of an exemplary peptide, ACA-PA.

FIG. 10F depicts bacterial viability using a live/dead assay forGram-negative MDR E. coli with 80 μM of an exemplary peptide, ACA-PA.

FIG. 11 depicts determination of LPS binding affinities of peptidesusing BC probe displacement assay.

FIG. 12A depicts TEM image of ultra-thin sectioned untreatedGram-positive MRSA.

FIG. 12B depicts TEM image of ultra-thin sectioned Gram-positive MRSAtreated with 40 μM of an exemplary peptide, ACA-PA.

FIG. 12C depicts TEM image of ultra-thin sectioned Gram-positive MRSAtreated with 80 μM of an exemplary peptide, ACA-PA.

FIG. 12D depicts TEM image of ultra-thin sectioned Gram-positive MRSAtreated with 80 μM of an exemplary peptide, ACA-PA.

FIG. 12E depicts TEM image of ultra-thin sectioned untreatedGram-negative MDR E. coli.

FIG. 12F depicts TEM image of ultra-thin sectioned Gram-negative MDR E.coli treated with 40 μM of an exemplary peptide, ACA-PA.

FIG. 12G depicts TEM image of ultra-thin sectioned Gram-negative MDR E.coli treated with 80 μM of an exemplary peptide, ACA-PA.

FIG. 12H depicts TEM image of ultra-thin sectioned Gram-negative MDR E.coli treated with 80 μM of an exemplary peptide, ACA-PA.

FIG. 13 depicts HDF cell viability after treatment with Bi-Cardinpeptide, CVK-PA or ACA-PA.

DETAILED DESCRIPTION

Overview

There are four major pathways to the development of bacterial drugresistance: i) alteration in the target site of antimicrobial agents toreduce binding affinity; ii) reducing drug accessibility via increasedefflux or decreased influx within the cell; iii) drug inactivation; andiv) tolerance that results in the survival of bacteria during inhibitedbacteria growth [2].

Antibiotic-resistance continues to be one of the biggest threats toglobal health. Antimicrobial peptides (AMPs) have been explored astherapeutic agents to treat antibiotic resistant microbes.Naturally-occurring AMPs can be found in many living organisms, wherethey serve as defense components in the innate immune system against abroad range of pathogens, including bacteria, viruses, and fungi [3-5].Unlike conventional antibiotics that mostly rely on receptor-specificpathways to display antibacterial activity, AMPs bind to negativelycharged bacteria cell membranes by non-specific physical interactionswith bacterial membranes, leading to cell death via membrane disruption.Moreover, AMPs possesses highly selective toxicity in bacteria comparedto mammalian cells, which is ascribed to the considerable differences inlipid composition between prokaryotic and eukaryotic cells. In bacterialmembranes, the negatively-charged hydroxylated phospholipids, such asphosphatidylglycerol (PG), cardiolipin (CL) and phosphatidylserine (PS),are abundantly present, while mammalian cell membranes are exclusivelyenriched by more electrically-neutral zwitterionic phospholipids andcholesterol that reduce peptide binding as well as membrane-thinningeffects of AMPs [6]. Although some AMPs isolated from natural sourceshave shown early success as alternatives to antibiotics, theirtherapeutic applications are limited by their high costs due to longsequences, limited supply, low stability to enzymatic degradation invivo, and off-target cytotoxicity [7, 8].

To overcome these challenges, sequence and structural features ofnaturally-occurring AMPs can be leveraged to develop strategies for thedesign of synthetic AMPs with wider therapeutic windows [9, 10].Characteristics of natural AMPs (such as cationicity, amphipathicity,and the ability to fold into secondary structures) can be retained insynthetic AMPs, which are key factors for their membrane permeation andmechanisms of antibacterial action. Most AMPs carry a positive netcharge from +2 to +9. Once the cationic domain of a peptide initiatesattachment with the anionic membrane surface, the hydrophobic domaindrives peptide partitioning into the non-polar regions of lipid bilayerto disrupt the membrane [6, 11]. Conjugation of cationic peptides withfatty acids [12] or hydrophobic amino acid residues [13] has been shownto meditate their antimicrobial activity. In addition, AMPs with β-sheetconformation can penetrate bacterial cell membranes via transmembranechannels and display potent antibacterial properties [7]. For instance,a series of short peptides that adopt β-sheet secondary structures onbacterial cell membranes have been shown to exhibit effectiveantibacterial properties against a broad range of clinically pathogenicbacteria strains, and possessed in vivo efficacy to treat fungalkeratitis [9, 14]. β-sheet forming short peptides that can form highlypacked nanorods showed enhanced antibacterial activity, which providessignificant evidence for the future of self-assembled peptides and theirclinical use [15].

A type of self-assembling peptide amphiphile (PA) has been designed tobe effective against antibiotic-resistant bacteria. Self-assembly ofthese PA systems into high aspect ratio and highly organizednanostructures is driven by non-covalent intermolecular interactions[18]. Cylindrical supramolecular structures of PA can be constructed byoligopeptide building blocks containing hydrocarbon chains, β-sheetforming groups via hydrogen bonding, and charged amino acids. Theseself-assembled nanostructures can be engineered for specific biologicalfunctions with a short peptide sequence displayed on the surface. Inrecent decades, PA systems have been extensively studied forapplications including regenerative medicines as well as drug deliveryvehicles.

Nonetheless, few studies have investigated the potential use ofself-assembling PA as therapeutics to treat drug-resistant bacteria.Here, amphiphilic Cardin-motif antimicrobial peptides (ACA-PA) weredesigned to self-assemble into cylindrical supramolecular structures.For example, this peptide self-assembly is driven by hydrophobicinteractions of the hydrophobic palmitic (16 carbon) tail groups, anddirected by the β-sheet forming V₄K₄ (SEQ ID NO: 1) peptide backbonesinto cylindrical shapes. The serial lysine groups act as a spacer andensure water solubility of the peptide amphiphiles [19, 20]. The ACA-PAwas functionalized with the heparin-binding (AKKARA)₂ (SEQ ID NO: 2)Cardin-Weintraub motif [21] to initiate attachment with the anionicbacterial cell membranes (FIGS. 1A and 1B). Self-assembling propertiesof the ACA-PA and the secondary structures were characterized.Antibacterial activity of the peptide was tested on Gram-positive MRSAas well as Gram-negative multi-drug resistant Escherichia coli (MDR E.coli), which possess resistance to beta-lactam antibiotics [24].Furthermore, mechanisms for the antibacterial activity, bacterialmembrane penetrating effects and cytotoxicity of ACA-PA were examined.

Self-Assembling Peptide Amphiphiles

Herein, self-assembling peptide amphiphiles (PAs) functionalized withheparin-binding Cardin-motifs (SEQ ID NO: 2, (AKKARA)₂, where A, K, andR represent alanine, lysine and arginine, respectively) have beendesigned to combat the drug-resistance of both Gram-positive andGram-negative bacteria. These cationic amphiphilic Cardin-motifantimicrobial (ACA) peptides could self-assemble in water into nanorods10 nm in diameter. Unlike typical small molecule antibiotics, theself-assembled ACA nanorods were shown to damage the bacterial cellmembrane and cause bacterial cell lysis. Such non-specific interactionscan reduce the development of drug-resistance, as the entire bacterialcell membrane is the target site of the self-assembled ACA nanorods. Incontrast, free (AKKARA)₂ peptides (SEQ ID NO: 2, without theself-assembly property) showed little antibacterial activity. Therefore,the unique self-assembled ACA nanorods with heparin-binding Cardin-motifsequences are promising antibacterial agents to treatantibiotic-resistant bacteria.

In some embodiments, PAs functionalized with the Cardin-motif canself-assemble into nanorods. In some embodiments, PAs functionalizedwith the Cardin-motif can self-assemble into nanofibers. In someembodiments, PAs functionalized with the Cardin-motif can self-assembleinto bundled and elongated nanofibers.

In some embodiments of the self-assembling peptide amphiphiles disclosedherein, the PA is a polypeptide comprising an amphiphilic backbone and acationic heparin-binding motif peptide. In some embodiments, theamphiphilic backbone comprises a hydrophobic portion and a beta-sheetforming segment.

In some embodiments of the self-assembling peptide amphiphiles disclosedherein, the polypeptide is represented by:R¹-y¹-y²wherein

R¹-y¹ is an amphiphilic backbone; and

y² is a cationic heparin-binding motif peptide.

In some embodiments, R¹ is an alkyl group or an alkenyl group. In someembodiments, R¹ is a C₁₀-C₂₂ alkyl group or a C₁₀-C₂₂ alkenyl group. Insome embodiments, R¹ is a C₁₂-C₂₂ alkyl group or a C₁₂-C₂₂ alkenylgroup. In some embodiments, R¹ is selected from the group consisting ofC₁₂, C₁₄, C₁₆, C16:1, C₁₈, C18:1, C18:2, C18:3, C₂₀, C20:1, C20:4,C20:5, C₂₂, C22:1, and C22:6. In some embodiments, R¹ is a C₁₆ alkylgroup.

In some embodiments, y¹ is a hydrophilic polypeptide. In someembodiments, y¹ has the following sequence (SEQ ID NO: 3):-(X_(n))-(Z_(n))-y³-wherein, independently for each occurrence:

X is I, L, or V;

Z is K or R;

n is 2, 3, 4, 5, 6, 7, 8, 9, or 10; and

y³ is absent, A, or G.

In some embodiments of y¹ disclosed herein, each X is I, L, or V. Insome embodiments, X is I. In some embodiments, X is L. In someembodiments, X is V.

In some embodiments, each Z is K or R. In some embodiments, Z is K. Insome embodiments, Z is R.

In some embodiments, each n is 2, 3, 4, 5, 6, 7, 8, 9, or 10. In someembodiments, n is 2. In some embodiments, n is 3. In some embodiments, nis 4. In some embodiments, n is 5. In some embodiments, n is 6. In someembodiments, n is 7. In some embodiments, n is 8. In some embodiments, nis 9. In some embodiments, n is 10.

In some embodiments, y³ is absent, A, or G. In some embodiments, y³ isabsent. In some embodiments, y³ is A. In some embodiments, y³ is G.

In some embodiments, y¹ has the following sequence V₄K₄ (SEQ ID NO: 1)or V₄K₄G (SEQ ID NO: 4).

In some embodiments, y² is a cationic heparin-binding motif peptide. Insome embodiments, y² has the following sequence (SEQ ID NO: 5):-(WZZWZW)_(m)wherein, independently for each occurrence:

W is A or G;

Z is K or R; and

m is 2, 3, 4, 5, 6, 7, 8, 9, or 10.

In some embodiments of y² disclosed herein, each W is A or G. In someembodiments, W is A. In some embodiments, W is G.

In some embodiments, each Z is K or R. In some embodiments, Z is K. Insome embodiments, Z is R.

In some embodiments, m is 2, 3, 4, 5, 6, 7, 8, 9, or 10. In someembodiments, m is 2. In some embodiments, m is 3. In some embodiments, mis 4. In some embodiments, m is 5. In some embodiments, m is 6. In someembodiments, m is 7. In some embodiments, m is 8. In some embodiments, mis 9. In some embodiments, m is 10.

In some embodiments, y² is -(AKKARA)_(m) (SEQ ID NO: 6).

In some embodiments, each of these PAs contains a hydrophobic portion(e.g., a C16 hydrocarbon group), a beta-sheet forming segment (e.g., SEQID NO: 1, V4K4), and a cationic heparin-binding functional group (e.g.,SEQ ID NO: 2, (AKKARA)₂).

In some embodiments of the PAs disclosed herein, the PA has thefollowing sequence C₁₆-V₄K₄G(AKKARA)₂ (SEQ ID NO: 7).

In some embodiments, the self-assembled nanorods are formed bynon-covalent forces including hydrogen bonds and hydrophobicinteractions, and display the cationic Cardin-motif on the surface.

In some embodiments of the polypeptides disclosed herein, thepolypeptide is incorporated into an article. In some embodiments, forexample, the article is selected from filters (e.g., hand-held waterfilters), membranes, packing materials (e.g., for foods, agriculture,paints, etc.), flow cells, filter gaskets, gloves, masks, garments,wound dressings, implants, catheters, and other medical devices. In someembodiments, the article is sterile.

Methods of Use

In some embodiments, PAs exhibited promising antibacterial properties.

In some embodiments, the addition of self-assembling peptide amphiphilicbackbones to cationic heparin-binding peptides significantly improvesthe antibacterial properties of the peptides, while the freeheparin-binding peptides only have little antibacterial effects.

In some embodiments, self-assembly of the PAs is important to theantibacterial effects. For example, once the nanorods areself-assembled, the bactericidal effects towards Gram-negative bacteriaare significantly more pronounced.

In some embodiments, the self-assembled ACA nanorods can be easilyprepared by dissolving the lyophilized peptides in water. Since theself-assembly is driven by non-covalent intermolecular interactions,complex chemical synthesis is not required for the preparation of thenanorods.

In some embodiments, these self-assembled ACA nanorods can kill a broadrange of bacteria including the antibiotic-resistant strains. Incontrast, many current antibiotics are only effective to eitherGram-negative or Gram-positive bacteria. For example, ciprofloxacin andofloxacin specifically target the DNA enzyme in Gram-negative bacteria,and moxifloxacin and gatifloxacin are more effective againstGram-positive bacteria.

In some embodiments, the cationic self-assembled nanorods can attach onthe negatively charged bacterial membranes by electrostaticinteractions, so the target side of the nanorods is the whole bacterialmembrane, which makes it more challenging for the bacteria to developresistance.

In some embodiments, ACA nanorods may cause the formation oftransmembrane pores on the surface of cell membranes, resulting incytoplasmic leakage followed by cell lysis.

Since peptide-based self-assembled materials are biodegradable, theseACA nanorods can be degraded after displaying their antibacterialeffects by proteolysis. Otherwise, undesirable retention of particles(such as synthetic polymeric and liposome materials) can cause systemictoxicity in liver, spleen and kidney.

In another aspect, the disclosure provides a method of antimicrobialtreatment, comprising:

providing a sample comprising a plurality of microorganisms;

adding to the sample a plurality of polypeptides comprising anamphiphilic backbone and a cationic heparin-binding motif peptide,thereby killing or inhibiting the growth of at least a portion of theplurality of microorganisms in the sample.

In some embodiments, at least a portion of the plurality ofmicroorganisms is killed. In some embodiments, the growth of at least aportion of the plurality of microorganisms is inhibited.

In another aspect, the disclosure provides a method of preventing orsuppressing microbial growth on a surface, comprising:

-   -   applying to the surface a plurality of polypeptides comprising        an amphiphilic backbone and a cationic heparin-binding motif        peptide, thereby preventing or suppressing microbial growth on        the surface.

In some embodiments, microbial growth on the surface is prevented. Insome embodiments, microbial growth on the surface is suppressed.

In some embodiments of the methods disclosed herein, the surface coatingis on an article. In some embodiments, for example, the article isselected from filters (e.g., hand-held water filters), membranes,packing materials (e.g., for foods, agriculture, paints, etc.), flowcells, filter gaskets, gloves, masks, garments, wound dressings,implants, catheters, and other medical devices. In some embodiments, thearticle is sterile.

In some embodiments of the methods disclosed herein, the sample furthercomprises water.

In some embodiments of the methods disclosed herein, the plurality ofpolypeptides comprising an amphiphilic backbone and a cationicheparin-binding motif peptide is any one of the self-assembling peptideamphiphiles disclosed herein. In some embodiments, the amphiphilicCardin-motif antimicrobial (ACA) peptides self-assemble.

In some embodiments, the microorganism is a bacterium, a virus, afungus, or a parasite. In some embodiments, the microorganism isantibiotic-resistant.

In some embodiments, the microorganism is a Gram-negative bacterium. Insome embodiments, the microorganism is a Gram-positive bacterium. Insome embodiments, for example, the microorganism is at least onebacterium selected from anthrax, Bacilli, Bordetella, Borrelia,botulism, Brucella, Burkholderia, Campylobacter, Chlamydia, cholera,Clostridium, Conococcus, Corynebacterium, diptheria, Enterobacter,Enterococcus, Erwinia, Escherichia, Francisella, Haemophilus,Heliobacter, Klebsiella, Legionella, Leptospira, leptospirosis,Listeria, Lyme's disease, meningococcus, Mycobacterium, Mycoplasma,Neisseria, Pasteurella, Pelobacter, plague, Pneumonococcus, Proteus,Pseudomonas, Rickettsia, Salmonella, Serratia, Shigella, Staphylococcus,Streptococcus, tetanus, Treponema, Vibrio, Yersinia and Xanthomonas. Insome embodiments, the microorganism is Staphylococcus aureus (S. aureus)or methicillin-resistant S. aureus (MRSA). In some embodiments, themicroorganism is Escherichia coli (E. coli) or multidrug-resistant E.coli (MDR E. coli).

In some embodiments, for example, the microorganism is at least onevirus selected from Adenoviridae, Papillomaviridae, Polyomaviridae,Herpesviridae, Poxviridae, Hepadnaviridae, Parvoviridae, Astroviridae,Caliciviridae, Picornaviridae, Coronoviridae, Flaviviridae,Retroviridae, Togaviridae, Arenaviridae, Bunyaviridae, Filoviridae,Orthomyxoviridae, Paramyxoviridae, Rhabdoviridae, and Reoviridae. Incertain embodiments, the virus may be arboviral encephalitis virus,adenovirus, herpes simplex type I, herpes simplex type 2,Varicella-zoster virus, Epstein-barr virus, cytomegalovirus, herpesvirustype 8, papillomavirus, BK virus, coronavirus, echovirus, JC virus,smallpox, Hepatitis B, bocavirus, parvovirus B19, astrovirus, Norwalkvirus, coxsackievirus, Hepatitis A, poliovirus, rhinovirus, severe acuterespiratory syndrome virus, Hepatitis C, yellow fever, dengue virus,West Nile virus, rubella, Hepatitis E, human immunodeficiency virus(HIV), human T-cell lymphotropic virus (HTLV), influenza, guanaritovirus, Junin virus, Lassa virus, Machupo virus, Sabia virus,Crimean-Congo hemorrhagic fever virus, ebola virus, Marburg virus,measles virus, molluscum virus, mumps virus, parainfluenza, respiratorysyncytial virus, human metapneumovirus, Hendra virus, Nipah virus,rabies, Hepatitis D, rotavirus, orbivirus, coltivirus, vaccinia virus,and Banna virus.

In some embodiments, for example, the microorganism is at least onefungus selected from Aspergillus (fumigatus, niger, etc.), Basidiobolus(ranarum, etc.), Blastomyces dermatitides, Candida (albicans, krusei,glabrata, tropicalis, etc.), Coccidioides immitis, Cryptococcus(neoformans, etc.), eumycetoma, Epidermophyton (floccosum, etc.),Histoplasma capsulatum, Hortaea werneckii, Lacazia loboi, Microsproum(audouinii, nanum etc.), Mucorales (mucor, absidia, rhizophus),Paracoccidioides brasiliensis, Rhinosporidium seeberi, Sporothrixschenkii, and Trichophyton (schoeleinii, mentagrophytes, rubrum,verrucosum, etc.).

In some embodiments, for example, the microorganism is at least oneparasite selected from Acanthamoeba, Babesia micron, Balantidium coli,Entamoeba hystolytica, Giardia lamblia, Cryptosporidium muris,Trypanosomatida gambiense, Trypanosomatida rhodesiense, Trypanosomabrucei, Trypanosoma cruzi, Leishmania mexicana, Leishmania braziliensis,Leishmania tropica, Leishmania donovani, Toxoplasma gondii, Plasmodiumvivax, Plasmodium ovale, Plasmodium malariae, Plasmodium falciparum,Pneumocystis carinii, Trichomonas vaginalis, Histomonas meleagridis,Secementea, Trichuris trichiura, Ascaris lumbricoides, Enterobiusvermicularis, Ancylostoma duodenale, Naegleria fowleri, Necatoramericanus, Nippostrongylus brasiliensis, Strongyloides stercoralis,Wuchereria bancrofti, Dracunculus medinensis, blood flukes, liverflukes, intestinal flukes, lung flukes, Schistosoma mansoni, Schistosomahaematobium, Schistosoma japonicum, Fasciola hepatica, Fasciolagigantica, Heterophyes heterophyes, and Paragonimus westermani.

Formulations

In another aspect, the disclosure provides a method of preparingself-assembled nanorods comprising the steps of:

dissolving a plurality of lyophilized polypeptides comprising anamphiphilic backbone and a cationic heparin-binding motif peptide in asolvent to form a mixture;

mixing the mixture; and

storing the mixture for a period of time to allow for supramolecularself-assembly, thereby preparing self-assembled nanorods.

In some embodiments, the solvent is selected from the group consistingof water, acetone, dimethylsulfoxide, ethanol, methanol, isopropanol,and mixtures thereof. In some embodiments, the solvent is water.

In some embodiments, the mixture is mixed by vortexing, shaking, oragitating. In some embodiments, the mixture is mixed by vortexing.

In some embodiments, the mixture is stored at a temperature below roomtemperature. In some embodiments, the mixture is stored at a temperaturebetween about −5° C. and about 25° C. In some embodiments, the mixtureis stored at a temperature between about 0° C. and about 20° C. In someembodiments, the mixture is stored at a temperature selected from thegroup consisting of about 20° C., about 19° C., about 18° C., about 17°C., about 16° C., about 15° C., about 14° C., about 13° C., about 12°C., about 11° C., about 10° C., about 9° C., about 8° C., about 7° C.,about 6° C., about 5° C., about 4° C., about 3° C., about 2° C., about1° C., and about 0° C. In some embodiments, the mixture is stored at atemperature of about 4° C.

In some embodiments, the mixture is stored for a period of time of atleast 1 h. In some embodiments, the mixture is stored for a period oftime of at least 5 h. In some embodiments, the mixture is stored for aperiod of time of at least 10 h. In some embodiments, the mixture isstored for a period of time of at least 15 h. In some embodiments, themixture is stored for a period of time of at least 20 h. In someembodiments, the mixture is stored for a period of time of at least 24h. In some embodiments, the mixture is stored for a period of time of atleast 30 h. In some embodiments, the mixture is stored for a period oftime of at least 36 h. In some embodiments, the mixture is stored for aperiod of time of at least 40 h. In some embodiments, the mixture isstored for a period of time of at least 45 h. In some embodiments, themixture is stored for a period of time of at least 48 h. In someembodiments, the mixture is stored for a period of time selected fromthe group consisting of about 1 h, about 2 h, about 3 h, about 4 h,about 5 h, about 6 h, about 7 h, about 8 h, about 9 h, about 10 h, about11 h, about 12 h, about 13 h, about 14 h, about 15 h, about 16 h, about17 h, about 18 h, about 19 h, about 20 h, about 21 h, about 22 h, about23 h, about 24 h, about 25 h, about 26 h, about 27 h, about 28 h, about29 h, about 30 h, about 31 h, about 32 h, about 33 h, about 34 h, about35 h, about 36 h, about 37 h, about 38 h, about 39 h, about 40 h, about41 h, about 42 h, about 43 h, about 44 h, about 45 h, about 46 h, about47 h, and about 48 h.

In some embodiments, the ACA nanorods in solution can be aimed to serveas antibacterial agents to treat eye infections. In some embodiments,the nanoparticles can be applied directly to the eye as eye drops insolution. In some embodiments, the nanoparticles in solution can be usedas a rinsing solution for contact lenses.

The building block peptides of the ACA nanorods can be efficientlyproduced by a cGMP process at a large scale with low cost. Bioprocessingthat produces peptides and proteins from recombinant DNA transfectedmammalian cells and bacteria has been widely used by many industrialpharmaceutical companies. Since the peptide amphiphiles of ACA nanorodsare short peptides, a gene that encodes this peptide sequence can betransfected into cells or bacteria, and the living organisms areincubated and able to generate the desired peptides exogenously.

Solid-phase peptide synthesis may also be used to produce largequantities of the peptides. Solid-phase peptide synthesis (SPPS),pioneered by Robert Bruce Merrifield, is the standard method forsynthesizing peptides and proteins in the lab. SPPS allows for thesynthesis of natural peptides which are difficult to express inbacteria, the incorporation of unnatural amino acids, peptide/proteinbackbone modification, and the synthesis of D-proteins, which consist ofD-amino acids.

Small porous beads are treated with functional units (‘linkers’) onwhich peptide chains can be built. The peptide will remain covalentlyattached to the bead until cleaved from it by a reagent, such asanhydrous hydrogen fluoride or trifluoroacetic acid. The peptide is thus‘immobilized’ on the solid-phase and can be retained during a filtrationprocess while liquid-phase reagents and by-products of synthesis areflushed away.

The general principle of SPPS is one of repeated cycles ofdeprotection-wash-coupling-wash. The free N-terminal amine of asolid-phase attached peptide is coupled to a single N-protected aminoacid unit. This unit is then deprotected, revealing a new N-terminalamine to which a further amino acid may be attached. The superiority ofthis technique partially lies in the ability to perform wash cyclesafter each reaction, removing excess reagent with all of the growingpeptide of interest remaining covalently attached to the insolubleresin.

Liquid-phase peptide synthesis is a classical approach to peptidesynthesis. It has been replaced in most labs by solid-phase synthesis;however, it retains usefulness in large-scale production of peptides forindustrial purposes.

Definitions

Unless otherwise defined herein, scientific and technical terms used inthis application shall have the meanings that are commonly understood bythose of ordinary skill in the art. Generally, nomenclature used inconnection with, and techniques of, chemistry described herein, arethose well-known and commonly used in the art.

The term “acyl” is art-recognized and refers to a group represented bythe general formula hydrocarbylC(O)—, preferably alkylC(O)—.

The term “acyloxy” is art-recognized and refers to a group representedby the general formula hydrocarbylC(O)O—, preferably alkylC(O)O—.

The term “alkoxy” refers to an alkyl group, having an oxygen attachedthereto. Representative alkoxy groups include methoxy, trifluoromethoxy,ethoxy, propoxy, tert-butoxy and the like.

The term “alkenyl”, as used herein, refers to an aliphatic groupcontaining at least one double bond and is intended to include both“unsubstituted alkenyls” and “substituted alkenyls”, the latter of whichrefers to alkenyl moieties having substituents replacing a hydrogen onone or more carbons of the alkenyl group. Typically, a straight chainedor branched alkenyl group has from 1 to about 20 carbon atoms,preferably from 1 to about 10 unless otherwise defined. Suchsubstituents may occur on one or more carbons that are included or notincluded in one or more double bonds. Moreover, such substituentsinclude all those contemplated for alkyl groups, as discussed below,except where stability is prohibitive. For example, substitution ofalkenyl groups by one or more alkyl, carbocyclyl, aryl, heterocyclyl, orheteroaryl groups is contemplated.

An “alkyl” group or “alkane” is a straight chained or branchednon-aromatic hydrocarbon which is completely saturated. Typically, astraight chained or branched alkyl group has from 1 to about 20 carbonatoms, preferably from 1 to about 10 unless otherwise defined. In someembodiments, the alkyl group has from 1 to 8 carbon atoms, from 1 to 6carbon atoms, from 1 to 4 carbon atoms, or from 1 to 3 carbon atoms.Examples of straight chained and branched alkyl groups include methyl,ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, tert-butyl, pentyl,hexyl, pentyl and octyl.

Moreover, the term “alkyl” as used throughout the specification,examples, and claims is intended to include both “unsubstituted alkyls”and “substituted alkyls”, the latter of which refers to alkyl moietieshaving substituents replacing a hydrogen on one or more substitutablecarbons of the hydrocarbon backbone. Such substituents, if not otherwisespecified, can include, for example, a halogen (e.g., fluoro), ahydroxyl, a carbonyl (such as a carboxyl, an alkoxycarbonyl, a formyl,or an acyl), a thiocarbonyl (such as a thioester, a thioacetate, or athioformate), an alkoxy, a phosphoryl, a phosphate, a phosphonate, aphosphinate, an amino, an amido, an amidine, an imine, a cyano, a nitro,an azido, a sulfhydryl, an alkylthio, a sulfate, a sulfonate, asulfamoyl, a sulfonamido, a sulfonyl, a heterocyclyl, an aralkyl, or anaromatic or heteroaromatic moiety. In preferred embodiments, thesubstituents on substituted alkyls are selected from C₁₋₆ alkyl, C₃₋₆cycloalkyl, halogen, carbonyl, cyano, or hydroxyl. In more preferredembodiments, the substituents on substituted alkyls are selected fromfluoro, carbonyl, cyano, or hydroxyl. It will be understood by thoseskilled in the art that the moieties substituted on the hydrocarbonchain can themselves be substituted, if appropriate. For instance, thesubstituents of a substituted alkyl may include substituted andunsubstituted forms of amino, azido, imino, amido, phosphoryl (includingphosphonate and phosphinate), sulfonyl (including sulfate, sulfonamido,sulfamoyl and sulfonate), and silyl groups, as well as ethers,alkylthios, carbonyls (including ketones, aldehydes, carboxylates, andesters), —CF₃, —CN and the like. Exemplary substituted alkyls aredescribed below. Cycloalkyls can be further substituted with alkyls,alkenyls, alkoxys, alkylthios, aminoalkyls, carbonyl-substituted alkyls,—CF₃, —CN, and the like.

The term “C_(x-y)” when used in conjunction with a chemical moiety, suchas, acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy is meant toinclude groups that contain from x to y carbons in the chain. Forexample, the term “C_(x-y) alkyl” refers to substituted or unsubstitutedsaturated hydrocarbon groups, including straight-chain alkyl andbranched-chain alkyl groups that contain from x to y carbons in thechain, including haloalkyl groups. Preferred haloalkyl groups includetrifluoromethyl, difluoromethyl, 2,2,2-trifluoroethyl, andpentafluoroethyl. C₀ alkyl indicates a hydrogen where the group is in aterminal position, a bond if internal. The terms “C_(2-y) alkenyl” and“C_(2-y) alkynyl” refer to substituted or unsubstituted unsaturatedaliphatic groups analogous in length and possible substitution to thealkyls described above, but that contain at least one double or triplebond respectively.

The term “alkylthio”, as used herein, refers to a thiol groupsubstituted with an alkyl group and may be represented by the generalformula alkylS—.

The term “amide”, as used herein, refers to a group

wherein each R^(A) independently represent a hydrogen or hydrocarbylgroup, or two R^(A) are taken together with the N atom to which they areattached complete a heterocycle having from 4 to 8 atoms in the ringstructure.

The terms “amine” and “amino” are art-recognized and refer to bothunsubstituted and substituted amines and salts thereof, e.g., a moietythat can be represented by

wherein each R^(A) independently represents a hydrogen or a hydrocarbylgroup, or two R^(A) are taken together with the N atom to which they areattached complete a heterocycle having from 4 to 8 atoms in the ringstructure.

The term “aminoalkyl”, as used herein, refers to an alkyl groupsubstituted with an amino group.

The term “aralkyl”, as used herein, refers to an alkyl group substitutedwith an aryl group.

The term “aryl” as used herein include substituted or unsubstitutedsingle-ring aromatic groups in which each atom of the ring is carbon.Preferably the ring is a 6- or 20-membered ring, more preferably a6-membered ring. The term “aryl” also includes polycyclic ring systemshaving two or more cyclic rings in which two or more carbons are commonto two adjoining rings wherein at least one of the rings is aromatic,e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls,cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls. Aryl groupsinclude benzene, naphthalene, phenanthrene, phenol, aniline, and thelike.

The terms “carbocycle”, and “carbocyclic”, as used herein, refers to asaturated or unsaturated ring in which each atom of the ring is carbon.Preferably, a carbocylic group has from 3 to 20 carbon atoms. The termcarbocycle includes both aromatic carbocycles and non-aromaticcarbocycles. Non-aromatic carbocycles include both cycloalkane rings, inwhich all carbon atoms are saturated, and cycloalkene rings, whichcontain at least one double bond. “Carbocycle” includes 5-7 memberedmonocyclic and 8-12 membered bicyclic rings. Each ring of a bicycliccarbocycle may be selected from saturated, unsaturated and aromaticrings. Carbocycle includes bicyclic molecules in which one, two or threeor more atoms are shared between the two rings. The term “fusedcarbocycle” refers to a bicyclic carbocycle in which each of the ringsshares two adjacent atoms with the other ring. Each ring of a fusedcarbocycle may be selected from saturated, unsaturated and aromaticrings. In an exemplary embodiment, an aromatic ring, e.g., phenyl, maybe fused to a saturated or unsaturated ring, e.g., cyclohexane,cyclopentane, or cyclohexene. Any combination of saturated, unsaturatedand aromatic bicyclic rings, as valence permits, is included in thedefinition of carbocyclic. Exemplary “carbocycles” include cyclopentane,cyclohexane, bicyclo[2.2.1]heptane, 1,5-cyclooctadiene,1,2,3,4-tetrahydronaphthalene, bicyclo[4.2.0]oct-3-ene, naphthalene andadamantane. Exemplary fused carbocycles include decalin, naphthalene,1,2,3,4-tetrahydronaphthalene, bicyclo[4.2.0]octane,4,5,6,7-tetrahydro-1H-indene and bicyclo[4.1.0]hept-3-ene. “Carbocycles”may be substituted at any one or more positions capable of bearing ahydrogen atom.

A “cycloalkyl” group is a cyclic hydrocarbon which is completelysaturated. “Cycloalkyl” includes monocyclic and bicyclic rings.Preferably, a cycloalkyl group has from 3 to 20 carbon atoms. Typically,a monocyclic cycloalkyl group has from 3 to about 10 carbon atoms, moretypically 3 to 8 carbon atoms unless otherwise defined. The second ringof a bicyclic cycloalkyl may be selected from saturated, unsaturated andaromatic rings. Cycloalkyl includes bicyclic molecules in which one, twoor three or more atoms are shared between the two rings. The term “fusedcycloalkyl” refers to a bicyclic cycloalkyl in which each of the ringsshares two adjacent atoms with the other ring. The second ring of afused bicyclic cycloalkyl may be selected from saturated, unsaturatedand aromatic rings. A “cycloalkenyl” group is a cyclic hydrocarboncontaining one or more double bonds.

The term “carboxy”, as used herein, refers to a group represented by theformula —CO₂H.

The term “ester”, as used herein, refers to a group —C(O)OR^(A) whereinR^(A) represents a hydrocarbyl group.

The term “ether”, as used herein, refers to a hydrocarbyl group linkedthrough an oxygen to another hydrocarbyl group. Accordingly, an ethersubstituent of a hydrocarbyl group may be hydrocarbyl-O—. Ethers may beeither symmetrical or unsymmetrical. Examples of ethers include, but arenot limited to, heterocycle-O-heterocycle and aryl-O-heterocycle. Ethersinclude “alkoxyalkyl” groups, which may be represented by the generalformula alkyl-O-alkyl.

The terms “halo” and “halogen” as used herein means halogen and includeschloro, fluoro, bromo, and iodo.

The terms “heteroaryl” and “hetaryl” include substituted orunsubstituted aromatic single ring structures, preferably 5- to20-membered rings, more preferably 5- to 6-membered rings, whose ringstructures include at least one heteroatom, preferably one to fourheteroatoms, more preferably one or two heteroatoms. The terms“heteroaryl” and “hetaryl” also include polycyclic ring systems havingtwo or more cyclic rings in which two or more carbons are common to twoadjoining rings wherein at least one of the rings is heteroaromatic,e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls,cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls. Heteroarylgroups include, for example, pyrrole, furan, thiophene, imidazole,oxazole, thiazole, pyrazole, pyridine, pyrazine, pyridazine, andpyrimidine, and the like.

The term “heteroatom” as used herein means an atom of any element otherthan carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen, andsulfur.

The terms “heterocyclyl”, “heterocycle”, and “heterocyclic” refer tosubstituted or unsubstituted non-aromatic ring structures, preferably 3-to 20-membered rings, more preferably 3- to 7-membered rings, whose ringstructures include at least one heteroatom, preferably one to fourheteroatoms, more preferably one or two heteroatoms. The terms“heterocyclyl” and “heterocyclic” also include polycyclic ring systemshaving two or more cyclic rings in which two or more carbons are commonto two adjoining rings wherein at least one of the rings isheterocyclic, e.g., the other cyclic rings can be cycloalkyls,cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls.Heterocyclyl groups include, for example, piperidine, piperazine,pyrrolidine, morpholine, lactones, lactams, and the like.

The terms “polycyclyl”, “polycycle”, and “polycyclic” refer to two ormore rings (e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, aryls,heteroaryls, and/or heterocyclyls) in which two or more atoms are commonto two adjoining rings, e.g., the rings are “fused rings”. Each of therings of the polycycle can be substituted or unsubstituted. In certainembodiments, each ring of the polycycle contains from 3 to 10 atoms inthe ring, preferably from 5 to 7.

The term “substituted” refers to moieties having substituents replacinga hydrogen on one or more carbons of the backbone. It will be understoodthat “substitution” or “substituted with” includes the implicit provisothat such substitution is in accordance with permitted valence of thesubstituted atom and the substituent, and that the substitution resultsin a stable compound, e.g., which does not spontaneously undergotransformation such as by rearrangement, cyclization, elimination, etc.Moieties that may be substituted can include any appropriatesubstituents described herein, for example, acyl, acylamino, acyloxy,alkoxy, alkoxyalkyl, alkenyl, alkyl, alkylamino, alkylthio, arylthio,alkynyl, amide, amino, aminoalkyl, aralkyl, carbamate, carbocyclyl,cycloalkyl, carbocyclylalkyl, carbonate, ester, ether, heteroaralkyl,heterocyclyl, heterocyclylalkyl, hydrocarbyl, silyl, sulfone, orthioether. As used herein, the term “substituted” is contemplated toinclude all permissible substituents of organic compounds. In a broadaspect, the permissible substituents include acyclic and cyclic,branched and unbranched, carbocyclic and heterocyclic, aromatic andnon-aromatic substituents of organic compounds. The permissiblesubstituents can be one or more and the same or different forappropriate organic compounds. For purposes of this invention, theheteroatoms such as nitrogen may have hydrogen substituents and/or anypermissible substituents of organic compounds described herein whichsatisfy the valences of the heteroatoms. Substituents can include anysubstituents described herein, for example, a halogen, a hydroxyl, acarbonyl (such as a carboxyl, an alkoxycarbonyl, a formyl, or an acyl),a thiocarbonyl (such as a thioester, a thioacetate, or a thioformate),an alkoxy, a phosphoryl, a phosphate, a phosphonate, a phosphinate, anamino, an amido, an amidine, an imine, a cyano, a nitro, an azido, asulfhydryl, an alkylthio, a sulfate, a sulfonate, a sulfamoyl, asulfonamido, a sulfonyl, a heterocyclyl, an aralkyl, or an aromatic orheteroaromatic moiety. In preferred embodiments, the substituents onsubstituted alkyls are selected from C₁₋₆ alkyl, C₃₋₆ cycloalkyl,halogen, carbonyl, cyano, or hydroxyl. In more preferred embodiments,the substituents on substituted alkyls are selected from fluoro,carbonyl, cyano, or hydroxyl. It will be understood by those skilled inthe art that substituents can themselves be substituted, if appropriate.Unless specifically stated as “unsubstituted,” references to chemicalmoieties herein are understood to include substituted variants. Forexample, reference to an “aryl” group or moiety implicitly includes bothsubstituted and unsubstituted variants.

The term “sulfonate” is art-recognized and refers to the group SO₃H, ora pharmaceutically acceptable salt thereof.

The term “sulfone” is art-recognized and refers to the group—S(O)₂—R^(A), wherein R^(A) represents a hydrocarbyl.

The term “thioether”, as used herein, is equivalent to an ether, whereinthe oxygen is replaced with a sulfur.

EXEMPLIFICATION

The invention now being generally described, it will be more readilyunderstood by reference to the following, which is included merely forpurposes of illustration of certain aspects and embodiments of thepresent invention, and is not intended to limit the invention.

Example 1: Preparation of the Self-Assembled Peptide AmphiphileSupramolecules

All of the peptides were synthesized by and purchased from Biomatik(Ontario, Canada). The peptides had purity >95% by HPLC. The sequencesof ACA peptide amphiphiles (ACA-PA) and Bi-Cardin peptides are listed inTable 1. For the self-assembling ACA-PAs, the self-assembly isunderstood to be driven by hydrophobic interactions of the hydrophobicpalmitic (16 carbon) tail groups, and also directed by the β-sheetforming V3K3 (SEQ ID NO: 8) peptide backbones into cylindrical shapes.With the rationally designed peptide backbones, the ACA-PAs canself-assemble into rod-like structures with cationic heparin-bindingCardin motifs exposed on the surface. ACA-PA contain palmitic tailgroups and β-sheet forming sequence with hydrophobic oligopeptideblocks, thereby allowing the peptide amphiphiles to self-assemble intoelongated structures with high aspect ratios. Heparin-binding Cardinmotif peptide sequence was incorporated in the C-terminus of the peptideto potentiate antibacterial activities against a broad spectrum ofbacteria.

To prepare the self-assembled supramolecules of the peptide amphiphiles,lyophilized ACA-PA powders were resuspended in deionized water (DIwater, Milli-Q system). Peptide amphiphile solutions were vortexed, andthen stored at 4° C. for at least 18 h to allow for supramolecularself-assembly. The solutions of the CVK peptide and Bi-Cardin peptidewere also prepared by the same method. In all the experiments, thepeptides were dissolved in autoclaved deionized water.

TABLE 1 Peptide Sequences and Molecular Weights of Exemplary Peptides.Peptide Molecular CMC Name Sequence Weight (Da) (μM) ACA-PAC₁₆-V₄K₄G(AKKARA)₂ 2475.28 45.7 (SEQ ID NO: 7) CVK-PA C₁₆-V₄K₄ 1165.6750.1 (SEQ ID NO: 1) Bi-Cardin (AKKARA)2 1269.57 N/A (SEQ ID NO: 2)

Example 2: Transmission Electron Microscopy (TEM) Characterization ofthe Self-Assembled Structures

The self-assembled structure of each PA was observed by TEM. As FIG. 2Ashows, the CVK-PA formed into uniform nanofibers with length in microns,and about 6 nm diameter. On the other hand, the self-assembled structureof ACA-PA can be influenced by concentration. At the concentration of 2mg/mL TEM revealed bundled and elongated nanofibers of ACA-PA (FIG. 2C),whereas nanorods with diameters from 7 to 10 nm were observed at theconcentration of 1 mg/mL (FIG. 2B). The Bi-Cardin peptide that solelycontains a heparin-binding group did not form any organized structure insolution.

Morphology characterization using TEM indicated that the formation oflong nanofibers for the CVK-PA measuring micrometers in length. TheACA-PA were also able to self-assemble into cylindrical supramolecularstructures but with relatively shorter lengths. Without wishing to bebound by any theory, the addition of cationic Cardin-motif groups to thepeptide backbone may have inhibited the formation of long nanofibers.Self-assembly is believed to be a result of a dynamic balance betweenthe hydrophobic interactions of the alkyl tail groups and the opposingrepulsive forces of the charged head groups. As the PAs aggregate andgrow into cylindrical structures, the electrostatic repulsion betweenlike-charged residues may limit the dimensions of the resultingsupramolecular structures [17, 26, 27, 29]. Interestingly, theself-assembled structures of ACA-PA exhibited morphology transition fromnanorod structures to bundle-like, longer fibers through the increase ofconcentration.

The self-assembled morphologies of peptide amphiphiles in water wereobserved by TEM (JEM-1010, Peabody, Mass.). Each sample was mounted on a300-mesh copper-coated carbon grid (Electron Microscopy Sciences,Hatfield, Pa.), and then negatively stained with a 1.5% phosphotungsticacid solution (1.5% PTA). Before imaging, all the samples were air-driedfor about 10 minutes. In all cases, samples were characterized at anaccelerating voltage at 80 kV.

Example 3: Circular Dichroism (CD) Measurements

As the circular dichroism spectra showed, the self-assembly of these ACAnanorods was directed by the β-sheet secondary structure which resultedfrom the designed peptide backbones, which plays an important role ininsertion of the nanorods into bacterial membranes.

In water, the CVK-PA adopted β-sheet secondary structure as FIG. 3Ashows a large positive peak at 203 nm and a negative peak at 218 nm. TheCD spectra of ACA nanorods underwent a blue-shift, which wascharacterized by a positive peak at 195 nm with reduced intensity and alarge negative peak at 208 nm (FIG. 3A). The CD spectra of the Bi-Cardinpeptide indicated the random-coiled structure characterized by anegative peak at around 198 nm (FIG. 3A). In the presence of prokaryoticmimicking lipid vesicles, all the peptide remained their secondarystructures as characterized in water (FIG. 3B). In membrane mimetic SDSsolution (25 mM), the CVK-PA were able to maintain the β-sheet secondarystructure, whereas the ACA-PA showed α-helix secondary structure withtwo negative peaks at 208 nm and 225 nm (FIG. 3C).

As characterized by CD measurements, spectra of the CVK-PA clearlyindicated a well-ordered β-sheet secondary conformation, which can beexerted by the formation of packed hydrophobic cores and β-sheet formingintermolecular hydrogen bonds. Regarding the ACA-PA, functionalizationof the cationic heparin-binding sequence caused a blue-shift on the CDspectra, leading to the spectra with 10 nm lower peak wavelengths andreduced ellipticity in the positive peak. Without wishing to be bound byany particular theory, this may be a consequence of increased positivelycharge and decreased peptide/peptide interactions [34]. Theconformational contribution induced by the Cardin-motif groups may beexcluded since: i) the Bi-Cardin peptide are unstructured in solution asmeasured by CD in this work, and ii) the free Cardin-motif peptidepossesses low α-helix content [23]. Investigation of peptide secondarystructures in lipidic environments suggests that both ACA-PA and CVK-PAcan maintain their conformational characteristics in prokaryoticmembranes. Interactions with the prokaryotic mimicking liposome vesiclesalso resulted in higher band intensity in the CD spectra of ACA-PA andCVK-PA, which can be attributed to peptide accumulation on the surfaceof anionic liposome vesicles [34]. It is also likely that the ACA-PA canexhibit transition to α-helix structure in a micelle-forming andmembrane-mimetic SDS solution.

The secondary structures of the peptides were characterized by a J-715spectropolarimeter (JASCO Analytical Instruments, Easton, Md., USA). The0.4 mM solutions of each peptide were prepared in degassed DI water.

To study the secondary structures of peptides under membrane-mimeticconditions, each peptide was suspended in either prokaryotic membranemimicking liposome vesicles or sodium dodecyl sulfate (SDS) solution(Thermo Fisher Scientific, Waltham, Mass., USA). Prokaryotic membranemimicking liposome vesicles were prepared as previously described [12].1,2-Dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE) and1,2-dilauroyl-sn-glycero-3-[phosphor-rac-(1-glycerol)] (DLPG) weredissolved in chloroform to a molar ratio of 7:3. A thin film of lipidswas formed by evaporating the solvent under vacuum at room temperaturefor 3 hours. The thin film was then rehydrated in DI water to aconcentration of 2.4 mM, followed by extrusion for 21 times with a 100nm Millipore polycarbonate filter. Structures of the resulting DLPE/DLPGliposome vesicles were confirmed by TEM characterization. Next, peptidesolutions were diluted in liposome vesicle solution or SDS solution. Thepeptide concentrations were fixed at 0.4 mM, whereas the finalconcentration of liposome vesicles and SDS solution were 0.3 mM and 25mM, respectively.

For CD measurements, 200 μL of each sample was transferred into a 1 mmpath length quartz cuvette (Hellma, Germany). The CD spectra werecollected between wavelengths of 190 nm to 240 nm at a scan rate of 10nm/min, and expressed as the average of three scans. All themeasurements were performed at room temperature.

Example 4: Determination of the Critical Micelle Concentration (CMC)

First, the solvatochromic probe Nile Red was used to examine thethreshold concentrations for the spontaneous aggregation induced byhydrophobic collapse of peptides. Once the supramolecular structures ofPA self-assemble in water at their CMC, the Nile Red dye can besolubilized in the hydrophobic cores of the PA assemblies, displaying anenhanced fluorescent intensity [28, 29]. Consequently, the fluorescentintensity maxima of Nile Red were at the emission wavelength of 630 nmin the solutions of CVK-PA and ACA-PA, whereas the emission wavelengthwas located at 660 nm in the Bi-Cardin peptide solutions (FIG. 4A-4D).The CMC of each peptide was also shown in Table 1 and FIG. 4A. BothCVK-PA and ACA-PA assembled at the concentrations of 45 μM and 50.1 μM,respectively, demonstrating the similar aggregation concentrations ofthese two types of PA. Without wishing to be bound by any theory, ACA-PAhave similar CMC as the CVK-PA, which may be the result of consonanthydrophobicity of the identical aliphatic tail groups. The Bi-Cardinpeptide did not induce such fluorescent enhancement for all theconcentrations tested, so it did not self-assemble.

The solvatochromic Nile Red dye(9-diethylamino-5-benzo[a]-phenoxazinone, Thermofisher, Waltham, Mass.,USA) was used to determine the CMC of each peptide. This dye is nearlynon-fluorescent in polar solvents but undergoes a blue shift emissiononce incorporated into hydrophobic environments. Briefly, the stocksolution of Nile Red (2.5 mM) was prepared in ethanol and then stored in−20° C. before use. 500 μL of the peptide solution ranging from 0.25 to300 μM were prepared by serial dilution. The dye solution was diluted by1,000-fold after 0.5 μL of the 2.5 mM Nile Red stock solution was placedinto 500 μL of each peptide solution. The solutions were then mixed, and100 μL of each solution were transferred onto a 96-well plate (eachsample was triplicate). The emission spectra of Nile Red in samples wascollected from 600 to 750 nm wavelengths at 5 nm intervals with anexcitation wavelength of 550 nm using a spectrophotometer (SpectraMaxM3, Molecular Devices, Molecular Devices, Sunnyvale, Calif., USA). Toestimate the CMC of each peptide, the corrected fluorescent intensity(I_(f, corrected)) of Nile Red at its emission wavelength for eachpeptide solution was calculated using equation:I_(f,corrected)=I_(f,sample)−I_(f,water), where I_(f, sample) is theaverage of the triplicate fluorescent intensity measurements in thesample and I_(f, water) is the fluorescent intensity of Nile Red inwater without the peptide. The measurements of the corrected fluorescentintensity were plotted against the log 10 values of concentration ofeach peptide, then the CMC value was determined at the point ofintersection of the plot of log peptide concentration and fluorescentintensity of Nile Red.

Example 5: Assessment of Bacterial Growth Inhibition

Gram-positive (S. aureus and MRSA) and Gram-negative (E. coli and MDR E.coli) bacteria were incubated with peptide concentrations ranging from20 to 100 μM, and time-lapsed bacterial growth was monitored for 20hours by determining the optical density at 562 nm (O.D. 562 nm). TheACA-PA remarkably inhibited the proliferation of both drug-susceptibleand drug-resistant bacteria, but demonstrated different inhibitoryeffects between Gram-negative bacteria and Gram-positive bacteria (FIGS.5A-5D). Gram-positive bacteria treated with ACA nanorods had a prolongedlag phase as the concentration increased. At the highest concentrationtested (100 μM), the time to exponential growth phase of S. aurues wasdelayed by about 18 h (FIG. 5B), and was prolonged by almost 13 h forMRSA (FIG. 5A), while bacteria incubated without ACA-PA entered theexponential growth phase within 4 h. Gram-positive bacteria were alsosusceptible to the ACA-PA even below their CMC.

On the other hand, peptide self-assembly was shown to be critical factorfor bacteria growth inhibition against Gram-negative E. coli and MDR E.coli bacteria. At concentrations higher than the CMC (60 μM to 100 μM),bacterial proliferation was significantly suppressed by the ACA-PA(FIGS. 5C and 5D). Specifically, no bacteria growth was observed for theMDR E. coli treated with ACA-PA over 80 μM, suggesting a bacteriostaticeffect was induced by the self-assembled nanorods. As such, ACA-PA atconcentrations lower than the CMC did not significantly inhibitbacterial growth. In addition, Bi-Cardin peptide was unable meaningfullyto inhibit the proliferation of either Gram-positive or Gram-negativebacteria at any of the concentrations tested (FIGS. 6A-6D). CVK-PApeptide showed some inhibition of the proliferation of at least MRSA atthe concentrations tested (FIGS. 7A-7D). The MIC values of ACA-PAtowards drug-resistant bacteria were determined after 24 h of incubation(Table 2). The MIC results demonstrated that the antibacterialactivities of ACA-PA were as effective as 78 U/mL and 625 U/mL ofpenicillin/streptomycin against MRSA and MDR E. coli, respectively.

TABLE 2 Minimum inhibitory concentration (MIC) values of ACA-PA todrug-resistant bacteria compared to penicillin-streptomycin (P/S).Bacterial Strain P/S ACA-PA MRSA  78 U/mL 300 μg/mL MDR E. coli 625 U/mL400 μg/mL

ACA-PA achieved a significantly stronger antibacterial effect, ascompared to CVK-PA with self-assembly backbone sequence and thenon-self-assembling Bi-Cardin peptide.

Without wishing to be bound by any theory, both heparin-binding groupsand β-sheet forming self-assembly groups may be important to theantibacterial properties of the PA, which could be explained by theenhanced cationic charge, hydrophobicity and the formation of β-sheetstructure of the ACA-PA. These physical and conformationalcharacteristics of the amphiphilic peptide promote binding to the lipidacyl chains of the membrane and enhance membrane insertion [33].Although the CVK-PA and Bi-Cardin peptide may interact with bacterialcell membranes via electrostatic interactions, the limited number ofcharged groups or hydrophobic residues may constrain their capabilityfor membrane insertion.

Interestingly, the Gram-positive (S. aureus and MRSA) and Gram-negative(E. coli and MDR E. coli) strains responded differently to the ACA-PAtreatment. Bacterial proliferation and the number of viableGram-positive bacteria decreased proportionally to the increasingconcentrations of ACA-PA. Even at concentrations below the CMC, the PAinhibited growth and promoted toxicity against the bacteria, whichimplied that the antibacterial property of the ACA-PA towardsGram-positive bacteria might not require peptide self-assembly. However,the results of bacterial studies towards Gram-negative bacteriaindicated the low potency of ACA-PA at the concentrations lower thanCMC. The dissociated PA had little antibacterial effect and, thus,peptide self-assembly was essential to kill Gram-negative bacteria. Onceself-assembled, the ACA nanorods considerably suppressed Gram-negativebacterial growth and induced a remarkable reduction in cell viability.

This assay was performed to investigate the inhibitory effects of eachpeptide against Gram-positive Staphylococcus aureus (S. aureus, ATCC#25923) and methicillin-resistant S. aureus (MRSA, ATCC #43300), as wellas Gram-negative Escherichia coli (E. coli, ATCC #25922), andmultidrug-resistant E. coli (MDR E. coli, ATCC #BAA-2471). In allexperiments, solutions of each peptide at the concentrations from 20 μMto 100 μM in 3% Tryptic Soy Broth (TSB) media were freshly prepared. Asingle colony of bacteria was isolated from a stock agar plate andcultured overnight in 4 mL of 3% TSB on a shaking incubator set at 200rpm and 37° C. The bacteria culture was adjusted to an optical densityat 562 nm (OD₅₆₂) to about 0.52 (10⁹ cells/mL), and the resultingbacteria suspension was subsequently diluted to about 10⁶ colony formingunits (CFU) per mL in each sample. The control samples were bacteriaincubated in TSB media without peptides. The bacteria were incubatedinside a spectrophotometer (Spectra-Max Paradigm, Molecular Devices,Sunnyvale, Calif.) at 37° C. under static conditions for 20 h, and thebacterial growth curves were measured in terms of turbidity at OD₅₆₂every 2 minutes [24]. The normalized OD₅₆₂ values of samples werecalculated by subtracting the experimental OD₅₆₂ values from thecorresponding blank samples without bacteria.

The minimum inhibitory concentration (MIC) of ACA-PA was tested againstdrug-resistant bacteria and compared with conventionalpenicillin-streptomycin (10,000 U/mL, Thermo Fisher Scientific, Waltham,Mass., USA) as well. Solutions of each antimicrobial agent at differentconcentrations were prepared by serial 2-fold dilutions in water.Bacteria (seeding density=10⁶ CFU/mL) were then mixed with theantimicrobial solutions, and incubated for 24 h at 37° C. The OD₅₆₂ wasmeasured at the end of treatment. The MIC value was determined as thelowest concentration of each antimicrobial agent that induced noincrease in OD₅₆₂. All experiments were run in triplicate to demonstratesignificance. Data are expressed as mean±standard error of the mean(S.E.M.) and a two-tailed Student t-test was used to evaluatedifferences between means, with p<0.005 being considered statisticallysignificant.

Example 6: Evaluation of Bactericidal Property

The bactericidal effects of the peptides were examined using a viablecolony count assay as well as the live/dead staining assay. For theviable colony count assay, bacteria were treated with peptides for 4 hand the resulting viable bacterial density of each sample was expressedby log₁₀[CFU/mL]. For the Gram-positive bacteria tested, the decrease inbacterial density was dependent on the concentration of the ACA-PA (FIG.8A). At concentrations over 80 μM, the ACA nanorods possessedsignificant bacteria toxicity which decreased colony forming units forboth S. aureus and MRSA by two logs, which suggested a 100-fold declinein bacteria density. As for Gram-negative E. coli and MDR E. colibacteria (FIG. 8B), the ACA nanorods, wherein the PA were added at aconcentration above their CMC, had potent bactericidal effects. Forexample, the ACA nanorods at concentrations over 60 μM reduced thecolony forming units of Gram-negative bacteria by about a five log unitreduction after 4 h compared with the untreated negative control sample.However, no significant reduction in Gram-negative bacteria density wasobserved in the samples treated with ACA-PA at concentrations lower thanCMC. These results indicated that the self-assembly of the ACA nanorodsenhanced the antibacterial activity against Gram-negative bacteria.Additionally, CVK-PA and Bi-Cardin peptide showed no bactericidaleffects towards Gram-positive or Gram-negative bacteria in comparison tothe ACA-PA. These results were consistent with the observations from thegrowth curves of the bacteria treated with PA.

In addition, ACA-PA were mixed with heparin at various weight ratios(1:1, 1:2, and 1:3 ACA-PA to heparin ratios) to block the Cardin motifgroups on the PA. The ACA-PA concentration used to treat bacteria (MRSAand MDR E. coli) was a constant 80 μM and only varied in theconcentration of heparin. The results indicated that the number ofviable bacteria colonies were positively proportional to the amount ofheparin added to the ACA-PA, although the self-assembled structure ofthe ACA nanorods was not affected by the addition of heparin (FIGS. 9Aand 9B). The ACA nanorods could be saturated at the 1:3 ACA-PA toheparin weight ratio as no toxicity towards bacteria was observed.Without wishing to be bound by any theory, these results suggested thatthe heparin-binding cationic Cardin-motifs participated in theantibacterial activity of the ACA nanorods.

The viable count assay was performed to quantify the bactericidalactivity of each peptide [25]. Stock solutions of peptides were dilutedin 3% TSB media to concentrations ranging from 20 μM to 100 μMimmediately before experiments. Heparin-ACA-PA were also prepared bymixing the ACA-PA stock solution with heparin (sodium salt from porcineintestinal mucosa, Sigma, St. Louis, Mo.) to 1:1, 1:2 and 1:3 weightratios in water, and a final concentration of ACA-PA in theheparin-ACA-PA at each heparin weight ratio was adjusted to 80 μM foreach experiment. The bacteria (10⁶ CFU/mL) were incubated with peptidesat 37° C. for 4 h. After incubation, serial dilutions of samples wereplated on TSB agar and then incubated at 37° C. overnight. The coloniesfrom each spot were manually counted, and the CFU number for each samplewas determined. Experiments were conducted in triplicate and resultswere expressed as the log 10 values of the CFU number in each sample.Data were expressed by stand error of the mean (±S.E.M.) and N=3,*p<0.05 compared with control samples and #p<0.05 compared with bacteriatreated with 40 μM of ACA-PA.

To further investigate bacteria viability after treatment with ACA-PA,the live/dead assay was used to confirm the antibacterial actions ofACA-PA. Dead bacteria with disrupted membrane were stained a firstcolor, whereas live bacteria were stained with a second colorfluorescence. A large amount of robust MRSA bacteria in the untreatedcontrol sample appeared a second color and in spherical shapes (FIG.10A). Followed by treatment with 40 μM of ACA-PA for 4 h, the increasein a first color fluorescent spots and decreased number of second colorfluorescent stained cells suggested markedly reduced MRSA viability(FIG. 10B). Interestingly, dead bacteria in a first color fluorescencetended to aggregate into bacterial clusters, which might result from theaggregation of de-stabilized bacterial cell membranes [25]. Furthermore,the coverage of dead bacteria increased with low density of livebacteria after being treated with 80 μM of ACA-PA (FIG. 10C). Theseresults further confirmed that ACA-PA were capable of inducingGram-positive bacterial cell death below their CMC. This antibacterialactivity was dependent on the concentration of PA, yet was notdetermined by peptide self-assembly. Nonetheless, Gram-negative MDR E.coli responded to the treatment of ACA-PA differently from Gram-positiveMRSA. The control MDR E. coli had a rod-shaped morphology (FIG. 10D).The treatment with 40 μM of ACA-PA was unable to induce considerablecell mortality (FIG. 10E). Above the CMC at 80 μM, the self-assembledACA-PA enhanced the number of dead bacteria drastically (FIG. 10F),indicating a prominent improvement in the antibacterial property of thePA against Gram-negative bacteria upon peptide self-assembly.

The live/dead bacterial viability assay was conducted to further assessthe bactericidal efficacy of ACA-PA using the LIVE/DEAD BacLightBacterial Viability Kits (Kits 71007, Molecular Probes, Eugene, Oreg.,USA). MRSA and MDR E. coli bacteria (100 μl, 10⁶ CFU/mL) were incubatedwith 80 μM and 40 μM of peptide in 3% TSB media at 37° C. for 4 h on a96-well plate, and the control samples of bacteria were incubated in 3%TSB media only. At the end of treatment, the supernatant of each samplewas carefully removed, and the bacteria on the bottom of the 96-wellplate were stained with reagent solution that was prepared according tothe manufacturer's instructions, and incubated at room temperature for10 minutes Samples were observed in a fluorescent microscope using AlexaFluor 488 (emission/excitation wavelength=500/519 nm) and PI (excitationemission=490/635 nm) filter sets. The live bacteria stained fluorescenta second color (SYTO9 nucleic acid stain), whereas the dead bacteriawith disrupted membranes stained fluorescent a first color (propidiumiodide, PI).

Example 7: Lipopolysaccharides (LPS) Binding Assay

The binding of each peptide at various concentrations to LPS wasdetermined by the capability of displacing the BC probes bound with LPS.The fluorescent BC probe was used as a probe to determine the level ofpeptide bound with LPS. BC probe binds to native LPS and particularlytarget the lipid A portion. The fluorescent intensity of BC probedecreases as it binds to LPS. Nonetheless, once the cationic peptidesthat that have competitively stronger interactions with LPS, BC probeson LPS are displaced and thus exhibit fluorescent enhancement [26, 27].As FIG. 11 reveals, all the peptides were able to replace the BC probeand caused enhanced fluorescent intensity of the probe compared tosamples without peptides, which demonstrated their interactions with thenegatively charged LPS. Moreover, the peptides displayed differentbinding activities with LPS. The LPS binding factor of Bi-Cardin peptideremained in similar levels and was irrespective of the increasingpeptide concentration. The CVK-PA showed weak binding affinity to LPS atconcentrations from 20 μM to 40 μM, but introduced significantlyenhanced binding affinity at concentrations higher than CMC. Forinstance, the LPS binding factor of samples containing 60 μM of CVK-PAwas as double as the 40 μM sample. The result indicated thatself-assembly of CVK-PA at concentrations over its CMC can improve itsinteractions with LPS. Furthermore, the ACA-PA exhibited the highest LPSbinding capability among the peptides investigated at all theconcentrations. Nonetheless, no statistically significant difference wasobserved between peptide concentrations below and higher than CMC,suggesting the strong interactions of ACA-PA may be irrelative topeptide self-assembly.

LPS is one of the major components in the outer membrane of mostGram-negative bacteria, which is considered the extra protection tobacteria. The core region of LPS can protect the organism fromhydrophobic antibiotics, whereas the mutations in porins can decreasethe entry of hydrophilic antimicrobial compounds and result in drugresistance. Thus, antibacterial agents that can penetrate anddestabilize LPS barrier can possibly inhibit resistance of bacteria[35]. To further investigate the interactions with LPS and mechanisms ofantibacterial actions towards Gram-negative bacteria, LPS bindingaffinity of each peptide was analyzed in this study. The results showedthat both Bi-Cardin peptide and CVK-PA can associate with LPS, althoughthese two peptides had low antibacterial activities as observed in thebacterial studies. Interestingly, the binding affinity of CVK-PA to LPSdramatically enhanced when it self-assembled into supramolecularnanofibers beyond the CMC at about 50 μM. These findings suggest thatself-assembly of peptide amphiphiles monomers into highly organizednanostructures can play a crucial role in association with LPS layer inbacteria, and also indicate the potential applications of incorporatingself-assembling properties in peptide-based antibiotics for improved LPSdestabilization. In addition, the ACA-PA exhibited the most effectiveLPS binding actions over all the concentrations investigated, which mayresult from the electrostatic interactions of the positively chargedpeptide with anionic LPS. Since the ACA-PA showed to have high LPSbinding affinity even at concentrations lower than CMC, theelectrostatic interactions of the peptide seemed to dominate over theeffects of peptide self-assembly on LPS binding. This can be explainedby the “self-promoted uptake pathway” in the interactions of bacterialLPS with cationic peptides. Peptides with strong affinities to LPS cancompetitively displace divalent cations (i.e. Ca²⁺ and Mg²⁺), resultingin disruption in the outer membrane. The antimicrobial peptides canfurther perpetrate into the cytoplasm membrane as the damaged outermembrane passively internalizes more molecules from the exterior [3,36]. In summary, we have found that the potent antibacterial propertiesof ACA-PA against Gram-negative bacteria can be ascribed to its strongLPS binding affinity, and the peptide self-assembly as well as thepositive charge may participate in the interactions with LPS.

The LPS binding affinity of peptide was examined with the BODIPY TRcadaverine (BC) fluorescent probe displacement assay as previouslydescribed [26, 27]. Stock solution of each peptide was diluted topredetermined concentrations with 10 mM HEPES buffer (pH=7.5, ThermoFisher Scientific, Waltham, Mass., USA). Peptide solutions wereequilibrated for 10 minutes, and LPS (from Escherichia coli O111:B4,Sigma, St. Louis, Mo., USA) solution in 10 mM HEPES buffer was added.The final peptide concentrations ranged from 20 μM to 100 μM, and theLPS concentration was 100 μg/mL. LPS only samples were solutions thatonly contained the same amount of LPS without peptide. Peptides wereallowed to interact with LPS in room temperature for 15 minutes, and theBC probe solution in 10 mM HEPES buffer was added to achieve a finalconcentration of 10 μM. Followed by incubation for 15 minutes in roomtemperature, the fluorescent intensity was measured using aspectrophotometer at an excitation wavelength of 580 nm and an emissionwavelength of 620 nm. Finally, the LPS binding factor of each sample wascalculated using the equation:

${{LPS}\mspace{14mu}{binding}\mspace{14mu}{factor}} = {1 - \frac{{{Fp} - {Fp}},{LPS}}{{Fp} - {FLPS}}}$

where Fp is the fluorescent intensity of BC probe in each peptidesolution, Fp,LPS is the fluorescent intensity of BC probe in the mixtureof peptide/LPS solution, FLPS represents the fluorescent intensity of BCprobe in solution containing LPS only.

When peptides bind to LPS and displace the BC probe, fluorescenceintensity of the probe is increased. LPS binding affinity of eachpeptide was then analyzed by the fluorescent intensity of the displacedBC probe. Data were expressed by stand error of the mean (±S.E.M.) andN=3, *p<0.05 compared with 0 μM samples and #p<0.05 compared with 40 μMsamples of the same peptide.

Example 8: Analysis of Bacterial Membrane Disruption Using TEM

TEM was used to reveal the damage in bacterial cell envelopes caused byACA-PA treatment. Drug-resistant bacterial strains (MRSA and MDR E.coli) were exposed to ACA-PA at concentrations below (40 μM) and above(80 μM) the CMC, and ultrathin sections of the bacterial cultures werevisualized by TEM. FIG. 12A shows proliferating, untreated MRSA with anintact peptidoglycan thick layer as well as a lipid membrane, ascharacterized by the formation of a cross wall [30]. In the cytoplasmicregion enclosed in the cell membrane, ribosomes appear in dark areaswith high electron density while the nucleoid displayed lower electrondensity [31, 32].

After treatment with 40 μM of ACA-PA, the thick layer of thepeptidoglycan envelope of MRSA was partially damaged and the innercytoplasmic membrane was no longer attached to the outer membrane,leading to cytoplasmic leakage (FIG. 12B), although the concentration ofPA was insufficient for self-assembly. The disintegration of thebacterial cell wall became apparent with increasing concentrations ofthe ACA-PA (FIGS. 12C and 12D), which demonstrated localized disruptionon the cell membrane and complete leakage of the cytoplasm. On the otherhand, the control Gram-negative MDR E. coli bacteria possess alipopolysaccharide (LPS) outer membrane that enclosed a thinpeptidoglycan membrane followed by a cytoplasmic inner membrane (FIG.12E). The 40 μM ACA-PA treatment caused blisters only on the outer cellwalls of the Gram-negative bacteria (FIG. 12F), but the bacterial innermembranes were intact and the cytoplasm was enclosed. The ACA-PA at aconcentration lower than CMC seemed to only influenced the outerbacteria membrane, but were inadequate to disrupt the inner bacteriamembranes. However, in FIGS. 12G and 12H, disrupted bacteria envelopeswith loosened and disconnected membranous structures were observed aftertreatment with 80 μM ACA nanorods.

Bacterial membrane disruption was observed from the ultrathin sectionedsample using TEM. The ACA-PA caused localized membrane disintegration onGram-positive MRSA at both 40 and 80 μM, further indicating that themembrane disruption effects were independent of peptide self-assembly.Nonetheless, ACA-PA at 40 μM (below the CMC) only caused preliminaryblisters on the outer membrane of Gram-negative bacteria and wereinadequate to induce complete membrane disruption as the cell membraneswere still able to enclose the cytoplasm. After treatment with 80 μM ACAnanorods, bacterial cell membranes were severely damaged with completecytoplasm leakage. Both outer and inner membranes demonstrateddisorganized structures throughout the bacterium. On the damagedmembranes, blisters appeared to originate from the pores. This explainsthe reason for the different antibacterial activity of ACA-PA onGram-negative bacteria compared to the concentration-dependent toxicityon Gram-positive bacteria. The cell envelope of Gram-positive has acytoplasmic membrane surrounded by a thick peptidoglycan layer withlipoteichoic acid. The antibacterial ACA-PA could penetrate theGram-positive bacterial membrane by charge and hydrophobicity, althoughthe PA were in a dispersed, non-assembled form. However, such dispersedPA were not able to penetrate the outer membrane of Gram-negativebacteria. Without wishing to be bound by any theory, it is proposed thatat concentrations higher than the CMC, ACA-PA induced bacterial celllysis possibly by the formation of transmembrane pores through the outerand inner membranes, which can be supported by the toroidal model. Inthe toroidal model, insertion of antibacterial peptides induces a localbending in the membrane leaflets through the pores, resulting inunfavorable elastic tension and ultimately lead to membranedisintegration [9]. Since the formation of self-assembled peptidenanoparticles can increase the local density of the cationic charge[10], once the self-assembly occurred at concentrations over CMC, it islikely that the self-assembled nanorods with localized charge and theirβ-sheet structure can insert into the cell wall layers perpendicularlyand form transmembrane pores, initiating membrane disintegration andcytoplasmic leakage.

To observe the effects of ACA-PA on the bacteria cell membranes, thetreated drug-resistant bacteria (MRSA and MDR E. coli) were fixed,sectioned and visualized by TEM using methods previously described [25].Briefly, MRSA and MDR E. coli bacteria (10⁷ CFU/mL) were treated by ACAnanorods at 40 μM and 80 μM in TSB media for 4 h at 37° C. (the controlsamples were bacteria grown in TSB media at the same condition). Thebacteria were centrifuged and then fixed with 2.5% glutaraldehyde in0.1M sodium cacodylate buffer overnight at 4° C. After rinsing with thesodium cacodylate buffer three times, bacteria pellets were post-fixedwith 1% osmium tetroxide for 2 h and dehydrated by gradient ethanolsolutions (30%, 50%, 70%, 85%, 90% and 100%). Samples were infiltratedwith squetol resin. The resins were polymerized at 55° C. for 24 h andthin-sectioned using an ultra-microtome (Reichert-Jung Ultracut E,Reichert Technologies, Buffalo, N.Y.). The sectioned samples weremounted on 200-hex mesh copper-coated carbon grids and stained by 1.5%uranyl acetate before imaging.

Example 9: Cytotoxicity Assays

To evaluate the cytotoxicity of the peptides, human dermal fibroblasts(HDF) were treated for 24 h, and cell viability was determined via MTSassays (FIG. 13). The ACA-PA had significantly lower toxicity towardsHDF cells than that observed towards bacteria. At concentrations from 20μM to 60 μM, the PA showed minimal cytotoxicity, and resulted in over50% HDF viability at the highest concentration tested at 100 μM.

However, given that CVK-PA as well as ACA-PA showed noticeable toxicitytowards HDF cells, biocompatibility of these peptide amphiphiles shouldbe taken into consideration for their potential applications in clinicaltrials. Similar observation was also demonstrated by Newcomb et al. thatnanofiber-forming peptide amphiphiles bearing β-sheet and serial lysineresidues can have strong cytotoxicity [37]. The disclosed peptidesshowed toxicity and inhibitory effects towards drug-resistant bacteria.

Furthermore, although the self-assembled ACA-PA also had a moderatecytotoxicity at high concentrations, it was also significantly moreeffective against bacteria at concentrations from 60 to 100 μM than HDFcells. At this concentration range, the ACA nanorods induced about a100-fold decrease in Gram-positive bacteria density, and a 10,000-folddecrease in Gram-negative bacteria density, but caused comparativelylower percentages of reduction (48%) in HDF cell viability. TheBi-Cardin peptide was shown to be non-cytotoxic at all theconcentrations tested. The CVK-PA critically decreased the cellviability at concentrations higher than 20 μM, which induced over 40% ofcell death.

Human dermal fibroblast cells (HDF, Detroit 551, ATCC #CCL-110) werecultured in Dulbecco's modified Eagle medium (DMEM; Fisher, Pittsburgh,Pa.) supplemented with 10% fetal bovine serum (FBS) and 1%penicillin/streptomycin for 24 h. All cells were cultured to 90%confluence in a 37° C., humidified, 5% CO₂/95% air environment. Cells atpassage numbers of 4-7 were used in these experiments.

The MTS cell viability assay kit (Promega, Madison, Wis., USA) was usedto determine cytotoxicity of the peptides. First, HDF cells were seededon a 96-well plate at a cell density of 5,000 cells/well (equal to15,385 cells/cm²), and were incubated overnight for cell adhesion. Afterovernight incubation, the cell medium in each well was replaced by 100μL of cell culture medium containing various concentrations of peptides(20 μM to 100 μM). The control samples were cells incubated in cellculture medium only. After 24 h, the treatment medium in each well wasremoved. The cells were washed with phosphate buffered saline (PBS,pH=7.4, Fisher, Pa., USA) and 100 μL of fresh cell culture medium wasadded to each well, followed by the addition of 20 μL of the MTSreagent. After the cells were incubated for 2.5 h, the absorbance wasmeasured at a wavelength of 490 nm by a spectrophotometer (SpectraMaxM3, Molecular Devices, Molecular Devices, Sunnyvale, Calif., USA). Toestimate the cell numbers in each well, a standard curve expressing thelinear correlation between different cell densities and opticaldensities (R²>0.95) was plotted, and the cell numbers were determinedwith this standard curve from the OD₄₉₀ value recorded in each sample.The cell viability of each sample was calculated by: Cellviability=[(cell density per mL of treated sample)/(cell density per mLof control sample)]×100%. Data were expressed by standard error of themean (±S.E.M.) and N=3, *p<0.05 compared with control samples.

A self-assembled ACA-PA functionalized with a heparin-binding (AKKARA)₂(SEQ ID NO: 2) Cardin-motif sequence was designed to form nanoparticleswith cylindrical shapes. At concentrations above the CMC (45 μM), theACA-PA were found to self-assemble into nanorods about 7 to 10 nm indiameter. The ACA-PA was shown to have excellent antibacterialproperties against both Gram-negative and Gram-positive bacteria incontrast to the CVK-PA and Bi-Cardin peptide. Antibacterial resultsdemonstrated for the first time that ACA-PA had aconcentration-dependent toxicity against Gram-positive bacteria (S.aureus and MRSA). For Gram-negative bacteria (E. coli and MDR E. coli),the ACA-PA possessed bactericidal effects only in self-assembled form,which indicated that self-assembly played a crucial role inantibacterial activity against Gram-negative bacteria. In all cases, theCVK-PA and Bi-Cardin peptide showed minimal antibacterial effect. Withits strong interactions with LPS layer and formation of secondarystructure on the prokaryotic membrane, the ACA-PA could target bacteriamembranes and result in localized membrane disintegration againstGram-positive bacteria and blisters on the surface of Gram-negativebacteria, causing bacterial cytoplasmic leakage. Functionalization witha self-assembled PA effectively enhanced the antibacterial activity ofthe cationic Cardin-motif peptide sequence, and the resultingself-assembled nanorods are promising agents to combat infectiousdiseases caused by bacteria drug-resistance.

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The contents of the articles, patents, and patent applications, and allother documents and electronically available information mentioned orcited herein, are hereby incorporated by reference in their entirety tothe same extent as if each individual publication was specifically andindividually indicated to be incorporated by reference. Applicantsreserve the right to physically incorporate into this application anyand all materials and information from any such articles, patents,patent applications, or other physical and electronic documents.

EQUIVALENTS

The invention has been described broadly and generically herein. Thoseof ordinary skill in the art will readily envision a variety of othermeans and/or structures for performing the functions and/or obtainingthe results and/or one or more of the advantages described herein, andeach of such variations and/or modifications is deemed to be within thescope of the present invention. More generally, those skilled in the artwill readily appreciate that all parameters, dimensions, materials, andconfigurations described herein are meant to be exemplary and that theactual parameters, dimensions, materials, and/or configurations willdepend upon the specific application or applications for which theteachings of the present invention is/are used. Those skilled in the artwill recognize, or be able to ascertain using no more than routineexperimentation, many equivalents to the specific embodiments of theinvention described herein. It is, therefore, to be understood that theforegoing embodiments are presented by way of example only and that,within the scope of the appended claims and equivalents thereto, theinvention may be practiced otherwise than as specifically described andclaimed. The present invention is directed to each individual feature,system, article, material, kit, and/or method described herein. Inaddition, any combination of two or more such features, systems,articles, materials, kits, and/or methods, if such features, systems,articles, materials, kits, and/or methods are not mutually inconsistent,is included within the scope of the present invention. Further, each ofthe narrower species and subgeneric groupings falling within the genericdisclosure also form part of the invention. This includes the genericdescription of the invention with a proviso or negative limitationremoving any subject matter from the genus, regardless of whether or notthe excised material is specifically recited herein.

We claim:
 1. A polypeptide, comprising an amphiphilic backbone, and acationic heparin-binding motif peptide, wherein the polypeptide isrepresented by:R¹−y¹−y² wherein R¹-y¹ is the amphiphilic backbone; y² is the cationicheparin-binding motif peptide; R¹ is an alkyl group or an alkenyl group;and y¹ has the sequence V₄K₄ (SEQ ID NO: 1) or V₄K₄G (SEQ ID NO: 4). 2.The polypeptide of claim 1, wherein R¹ is a C₁₀-C₂₂ alkyl group or aC₁₀-C₂₂ alkenyl group.
 3. The polypeptide of claim 1 having the sequenceC₁₆ alkyl-V₄K₄G(AKKARA)₂ (SEQ ID NO: 7).
 4. The polypeptide of claim 1,wherein y² has the sequence (SEQ ID NO: 5):−(WZZWZW)_(m) wherein W is A or G; Z is K or R; and m is 2, 3, 4, 5, 6,7, 8, 9, or
 10. 5. The polypeptide of claim 4, wherein R¹ is a C₁₀-C₂₂alkyl group or a C₁₀-C₂₂ alkenyl group.
 6. The polypeptide of claim 4,wherein W is A.
 7. The polypeptide of claim 6, wherein R¹ is a C₁₀-C₂₂alkyl group or a C₁₀-C₂₂ alkenyl group.
 8. The polypeptide of claim 4,wherein Z is K.
 9. The polypeptide of claim 8, wherein R¹ is a C₁₀-C₂₂alkyl group or a C₁₀-C₂₂ alkenyl group.
 10. The polypeptide of claim 4,wherein y² has the sequence −(AKKARA)_(m)(SEQ ID NO: 6).
 11. Thepolypeptide of claim 10, wherein R¹ is a C₁₀-C₂₂ alkyl group or aC₁₀-C₂₂ alkenyl group.
 12. A method of antibacterial treatment,comprising: providing a sample comprising bacteria; adding to the samplea plurality of polypeptides of claim 1, thereby killing or inhibitingthe growth of at least a portion of the bacteria in the sample.
 13. Amethod of preventing or suppressing bacterial growth on a surface,comprising: applying to the surface a plurality of polypeptides of claim1, thereby preventing or suppressing bacterial growth on the surface.14. A method of preparing self-assembled nanorods comprising the stepsof: dissolving a plurality of lyophilized polypeptides of claim 1 in asolvent to form a mixture; mixing the mixture; and storing the mixtureto allow for supramolecular self-assembly, thereby preparingself-assembled nanorods.